23 December 2009. Research on epigenetic effects—heritable changes in gene expression caused by mechanisms, such as methylation, that do not alter DNA sequences—is in its very early days in psychiatry. However, epigenetics is attracting increasing interest among researchers because epigenetic research has suggested intriguing mechanistic links between environmental factors and lasting changes in cognition, behavior, and physiology (see, e.g., Zhang and Meaney, 2010).

Explorations of epigenetics in schizophrenia research are still few and far between (see SRF related news story; SRF news story; SRF news story), but a new study from Paul Greengard's and Alexander Tarakhovsky’s groups at The Rockefeller University of a mental retardation-like phenotype in mice may be a harbinger of like studies in schizophrenia and mood disorders in the coming decade (Renthal and Nestler, 2009).

The technique employed in the new work—a conditional, forebrain-specific, postnatal ablation of GLP/G9a, a histone methyltransferase complex that is crucial for epigenetic gene silencing during normal neural development—is analogous to the alteration of the GLP gene seen in the human 9q Subtelomeric Deletion Syndrome (9qSTDS), and the researchers observed phenotypes in the GLP/G9a-deficient mice that are strikingly similar to those seen in 9qSTDS patients.

A familiar phenotype
9qSTDS is associated with obesity, reduced muscle tone and motor activity, and a gradual decline in goal-directed behaviors and interest in the environment, leading to severe apathy. As reported by co-first authors Anne Schaefer and Srihari Sampath, at about six to eight weeks of age, the GLP/G9a-deprived mice in the new research showed significant declines in exploration and motor activity compared to their normal littermates, and they also showed significant deficits in fear conditioning. By six months of age, GLP/G9a-deprived mice weighed twice as much as controls. GLP/G9a-deprived mice showed virtually none of the marked preference for sucrose over plain water seen in normal mice.

Remarkably, these behavioral phenotypes were not accompanied by any obvious changes in forebrain cell morphology or neuronal architecture: the organization and structure of the hippocampus, striatum, and cortex appeared normal; granule cells, medium spiny neurons, and pyramidal cells were similar to those in control animals.

However, in the hippocampus, striatum, and cortex the researchers found a marked reduction in GLP-positive cells in GLP/G9a-deprived mice, as well as the abnormal presence of non-neuronal cells, whose development in these regions would normally be repressed by methylation effects of GLP/G9a. Immunofluorescence analysis of forebrain neurons confirmed that GLP/G9a methylation was largely abolished in euchromatic DNA, and some 60 genes, including many involved in the differentiation of non-neuronal cells, were found to be upregulated in the forebrain of GLP/G9a-deprived mice, compared to normal littermates.

In separate experiments, the authors specifically ablated GLP/G9a only in forebrain neurons expressing the D1 or D2 dopamine receptor. They did not observe the behavioral phenotypes seen in mice where GLP/G9a had been globally ablated in the forebrain, but did see marked increases in locomotor and exploratory behavior when these mice were treated with either a D1 agonist or with caffeine (which reduces D2 activity by acting as an antagonist at the adenosine-2-α receptor.

The authors attribute the mental retardation-like syndrome they observed to a widespread disruption in forebrain neurons caused by the expression of many non-neuronal genes (or early neuron progenitor genes) unleashed by GLP/G9a deficiency. More narrow disruption, such as that caused in those mice in which GLP/G9a was ablated only in dopaminergic neurons, they argue, may only be revealed by cell type-specific (in this case, pharmacological) stimuli.

“Epigenetic regulators govern expression of large numbers of unrelated genes,” the group writes. “Therefore, it is conceivable that mental retardation is triggered not by changes in specific target gene(s), but by the inability of neurons to respond adequately to environmental signals under conditions of greatly distorted transcriptional homeostasis.”—Pete Farley.

The paper by Mill et al. is one of the first comprehensive attempts to examine changes in methylation across the entire genome in patients with various diagnoses of mental illness. The study is well designed, extensive, and uses fairly new technology to examine changes in methylation profiles across the genome. In the frontal cortex, the authors provide evidence for psychosis-associated differences in DNA methylation in numerous loci, including those involved in glutamatergic and GABAergic transmission, brain development, and other processes linked with disease etiology. Methylation in the frontal cortex of the BDNF gene is correlated with a non-synonymous SNP previously associated with major psychosis. These data provide further support for an epigenetic origin of major psychosis, as evidenced by DNA methylation-induced changes likely important to gene expression.

In many ways, this seems reminiscent of the trend in genetics several years ago when the inclination was to move from single gene loci association and linkage studies to genomewide scans. The only downside of the approach is that what one gains in information, one (at least initially) loses in biology. That is given the wealth of new findings uncovered; we now need to go back and examine these results in light of what we know regarding gene function in neurobiology and cognition. Of course, this is the trend, now that microarrays have increased our capacity to look at all things at the same time. The flipside is that it will take several large-scale studies of this sort to better understand which findings are replicable and which are not. That is, do the results of the Mill paper agree with data obtained and carried out by laboratories using the methyl DIP or MeCP2 ChIP assays coupled with microarrays. While these experiments ask different questions, the implication is that there may be some degree of overlap in comparing these different methodologies. While this may be premature, there is a sense that this information will be available shortly.

Finally, I would like to focus on recent findings regarding the methylation of the reelin promoter. These authors (Mill et al.) and Tochigi and colleagues (Tochigi et al., 2008) have found that the reelin promoter is not hypermethylated in patients with schizophrenia. In fact, Tochigi et al., 2008, found that the reelin promoter is not methylated at all using pyrosequencing. However, several groups (Grayson et al., 2005; Abdolmaleky et al., 2005; Tamura et al., 2007; Sato et al., 2006) have shown that the human reelin promoter is methylated in different circumstances. Interestingly, there is little consensus in the precise bases that are methylated in these latter studies. Our group (Grayson et al., 2005) performed bisulfite treatment of genomic DNA and sequencing of individual clones. Moreover, we analyzed two distinct patient populations. The clones were sequenced at a separate facility. What was intriguing was that the baseline methylation patterns in the two populations was different, and yet several sites stood out as being relevant in both. We mapped methylation to the somewhat rare CpNpG sites proximal to the promoter. Interestingly, these bases were located in a transcription factor-rich portion (Chen et al., 2007) of the promoter and in a region that shows 100 percent identity with the mouse promoter over a 45 bp stretch. We have also been able to show that changing one of these two bases to something other than cytosine reduces activity 50 percent in a transient transfection assay. So the question becomes, How do we reconcile these disparate findings regarding methylation? As suggested by Dr. McCaffrey, the answer may lie in regional differences that arise due to the nature of the material available for each study. We have found a degree of reproducibility by using human neuronal precursor (NT2) cells for many of our studies. At the same time, this cell line is somewhat artificial and cannot be used to reconcile differences found in human tissue. Perhaps it might be prudent to examine material taken by using laser capture microdissection to enrich in more homogenous populations of neurons/glia. In moving ahead, it might be best to now focus on the mechanism for these differences in methylation patterns and try to understand the biology associated with the new findings (Mill et al., 2008) as a starting point.

The methylation difference between twins is clearly demonstrated using newer methods in this publication. However, conceptually it’s an old story now. A quick PubMed search for "monozygotic twins and non-identical" yielded a total of 7,653 publications. There is no doubt that the more we look, the more difference we will find between monozygotic twins. Also, monozygotic twin differences in methylation and gene expression are expected to increase with age. It is also affected by a variety of genetic and environmental factors. We have come a long way in genetic research on twins and the time has come to modify our thinking about monozygotic twins as "non-identical but closest possible" rather than as "identical." They started from a single zygote, but have diverged during development and differentiation including upbringing.

The implication of the published results is that the methylation (epigenetic) differences (in monozygotic twins) will be powerful in any genetic analysis of disease(s). Once again, it is probably more problematic than usually assumed. Also, it is particularly problematic for behavioral/psychiatric disorders including schizophrenia. The reason is multi-fold and includes the effect of (known and unknown) environment including pregnancy, upbringing, drugs, life style, food, etc. All these are known to affect DNA methylation and gene expression. As a result, they add unavoidable confounding factors to the experimental design. It does not mean that epigenetics is not involved in these diseases. Rather, directly establishing a role for methylation in schizophrenia will be challenging. A special limitation is the fact that methylation is known to be cell-type specific, and perfectly matched affected and normal (twin) human brain (region) samples for necessary experiments are problematic and methylation studies on other cell types may or may not be informative.